A vaccinia virus mutant which is resistant to aphidicolin (AP) codes for an AP-resistant DNA polymerase. A change from wild-type (WT) cytosine to adenine at position 2430 of the DNA sequence changes leucine to methionine in the polymerase and confers AP resistance. The mutation site is in a region of six amino acids which are conserved in several DNA polymerases. I am substituting other amino acids for the WT leucine at the original mutation site and testing for AP resistance. To proceed efficiently, I have developed the technique for site-directed mutagenesis described by Eckstein. This procedure produces recombinant single stranded (ss) DNA containing a mutation which is specified in an oligonucleotide primer. Frequently over 75% of the plaques produced by transformation contain the desired alteration so that a preliminary screen for the mutation is unnecessary. Also, one can anneal different primers with different substituted nucleotides to a single template. One reaction used three primers with different substitutions and only five plaques had to be picked to obtain the three mutations. Furthermore, sequence permutations over a short region can be amplified by annealing a single primer with substitutions at positions starting near one end and extending to the center of the oligonucleotide. Because centrally located alterations are obtained more frequently than peripheral ones, different plaques contain different sequence alterations. Also, the marker transfer reaction was modified to use ss recombinants rather than the usual plasmid (ds) DNA. Eight micrograms of ss DNA produced as many mutant plaques as one microgram of ds DNA. Finally, the template used for one set of AP mutagenic reactions contained the phosphonoacetate (PA) resistance mutation. This construction can be used to indicate whether or not a given substitution at the AP site is liable to be lethal since the recombinant can be tested for PA resistance. The results so far indicate that substitution of leucine by proline, arginine, tryptophan or glutamic acid does not confer AP resistance.